- Volume 18, Issue 3, 1973

Pencillium stoloniferum virus S has been fractionated into four particle classes, E, M, L and H, with sedimentation coefficients of 66, 87, 101 and 113 S, respectively. E particles were shown to be empty capsids, while M particles contained single-stranded RNA, L particles contained double-stranded RNA, and H particles contained both single-stranded RNA and double-stranded RNA. The mol. wt. of L particles was found from sedimentation and diffusion coefficients to be 6.0 × 106.

Evidence is presented that the M, L, and H particle classes each contain two components, M 1, M 2, L 1, L 2, H 1 and H 2, respectively. It has been shown that the M 1, M 2, L 1 and L 2 particles each contain only one molecule of RNA with mol. wts. of 0.47 × 106, 0.56 × 106, 0.94 × 106 and 1.11 × 106, respectively, while H 1 and H 2 particles each contain one molecule of double-stranded RNA of mol. wts. 0.94 × 106 and 1.11 × 106, respectively, together with single-stranded RNA.

Interferon-treated cells exhibited an increased susceptibility to the toxicity of several natural and synthetic double-stranded RNA molecules and to vaccinia virus, provided the latter was able to undergo replicative events. However, interferon-treated cells exhibited no enhanced susceptibility to several other toxic materials or to vaccinia virus that was unable to replicate. These findings suggest that interferon-treated cells exhibit a specific enhanced susceptibility to the toxicity of double-stranded RNA molecules. Since neither RNA nor protein synthesis was required for toxicity to occur in interferon-treated cells exposed to polyriboinosinic-polyribocytidylic acid, it appears that the double-stranded RNA configuration is directly lytic for interferon-treated cells.

Maternal antibodies to Sendai virus appeared to be transferred to newborn mice to a greater extent via colostrum and gastro-intestinal tract than via the placenta. Serum antibody reached a maximum within a week of birth and remained high for a few weeks before decreasing gradually. The immunoelectrophoretic analysis of serum globulins of suckling mice demonstrated IgG1 and IgG2 but no IgA, and suggested a selective transport of maternal globulins through the gastrointestinal epithelium of suckling mice. The maternal antibody effectively protected one-day-old newborn mice against intranasal challenge by a lethal dose of Sendai virus, but was less effective against the contact infection of 3-week-old mice. Thus, passive immunity appeared to be brief. Enzootic infections with Sendai virus are discussed on the basis of these experiments.

Radioisotope labelling, autoradiography, electron microscopy and DNA-DNA hybridization have been used to analyse the DNA species produced in a stable line of monkey kidney cells after infection with defective adeno-associated satellite virus (ASV), in presence of and without simian helper adenovirus (SV 15). Extraction of DNA by the Hirt (1967) procedure and CsCl equilibrium density gradient sedimentation indicated that a newly synthesized low mol. wt. DNA was readily isolated from cells infected with the defective satellite virus alone, within 6 h after inoculation. Sedimentation analyses of this DNA through neutral sucrose gradients showed that it was heterogeneous, with a sedimentation coefficient in the range 11 to 23 S. The DNA was non-infectious when titrated with helper adenovirus. Sedimentation analyses of DNA extracted from cultures infected with satellite virus and helper adenovirus showed linear 32 S adenovirus DNA and a 26 S linear DNA which may represent a replicative intermediate of satellite virus. Electron microscopy showed that the latter DNA was linear with marked propensity for clumping and concatenation. Molecules with side chains were seen. Autoradiography showed that newly synthesized DNA isolated from cells infected with satellite alone was nuclear in origin and DNA-DNA hybridization indicated that the synthesized DNA not extracted by the Hirt procedure was cellular rather than virus DNA.

Young, maturing and aged tobacco leaves were infected with tobacco mosaic virus (TMV) strain vulgare or flavum. TMV-RNA synthesis and accumulation were followed. Flavum and vulgare RNAs had different patterns of synthesis and accumulation: flavum-RNA may be unstable.

In healthy leaves, ribosomal RNA synthesis (measured by [32P] incorporation) increased to a peak before the leaf reached its maximum length then declined to 25% of the maximum as the leaf aged. TMV infection of a young leaf caused immediate and persistent inhibition of ribosomal RNA synthesis. Ribosomal RNA synthesis in older leaves showed three phases after TMV infection. (1) One day after inoculation, ribosomal RNA synthesis was higher than in healthy leaves. (2) During the main accumulation of TMV-RNA, ribosomal RNA synthesis was inhibited. (3) Following TMV-RNA accumulation, ribosomal RNA synthesis rose, often to levels higher than in healthy leaves. The half-life of ribosomal RNA in a TMV-infected leaf was found to be twice that in a healthy leaf.

These observations are discussed in relation to leaf development and virus multiplication.

When purified preparations of the cam isolate of tobacco rattle virus were stored the protein in the virus particles underwent limited proteolysis. Using polyacrylamide gel electrophoresis, a change was observed in the mol. wt. of the virus polypeptide from 28 500 to 23 500, and in some instances a possible intermediate of 26 000 mol. wt. was detected. Polypeptides of mol. wt. 13 500, 11 500 and 9000 were also detected after prolonged storage. Virus protein was not degraded when virus preparations containing sodium azide or antibiotics were stored, but it was degraded in the presence of azide when incubated with the supernatant fluid obtained by centrifuging preparations of virus which had been stored for several months without bacteriostatic additive. The proteolysis was probably caused by microbial exoenzyme(s). The conversion of polypeptide from mol. wt. 28 500 to 23 500 did not affect sedimentation coefficient, behaviour when centrifuged to equilibrium in caesium chloride, appearance in the electron microscope, serological reactivity, infectivity, heat stability or resistance of infectivity to pancreatic ribonuclease.

Purified virus was not affected by treatment with trypsin or chymotrypsin whereas the virus polypeptide was degraded by pronase and by papain. Papain had effects similar but not identical to those of storage. These enzyme treatments did not greatly affect infectivity or alter the appearance of virus particles in the electron microscope.

Isolate prn was affected by storage and by papain treatment in the same way as isolate cam, but more slowly.

The production, purification and properties of the ‘A’ antigen induced by Marek’s disease virus in tissue culture were investigated. It was found that although virus multiplied on passage in chick kidney, chick embryo and duck embryo cells the antigen was produced only in chick kidney and duck embryo cultures. The results suggested that the ‘A’ antigen produced by duck embryo cells was a glycoprotein having a mol. wt. in the range 70 000 to 90 000 and was heterogeneous in charge (pI 4.5 to 5.5). A 20-fold purification was achieved by electrophoresis in 5% acrylamide gel with a recovery of 45%. An equally good purification was also possible by chromatography on DEAE-Sephadex A50 and Sephadex G200, although the recovery was only 20% in this case.

Treatment of purified Newcastle disease virus (NDV) with Triton N101 to activate the virus particle RNA polymerase in the in vitro assay removed the glycoproteins and most of the phospholipid from the virus. The subvirus particles produced by the action of Triton N101 on virus had RNA polymerase activity, whereas purified NDV nucleocapsids did not. Apparently, either nucleocapsids did not contain the proteins essential for RNA polymerase activity, or the enzyme was denatured during the preparation of nucleocapsids. Incubation of several purified strains of NDV at 4 °C led to loss of polymerase activity which was regained on incubation at 37 °C. It is suggested that internal configurational changes in the virus particle were the most likely causes of these changes in enzyme activity on incubation at 4 °C and 37 °C.

A direct correlation was found between the affinity of certain cardioactive sterols for the binding site of (Na+, K+)-dependent ATPase and their capacity to raise poliovirus yields in single cycles of infection in suspended monkey kidney cells. The latter effect was due to an improved efficiency of infection by the attached particles rather than to higher yields/cell.

The average number of p.f.u. attached/cell within a certain time was directly proportional to the number of free p.f.u. initially present. In untreated systems, the efficiency of initiation of infection was low and was not directly related to the average number of p.f.u. attached/cell. The addition of digitoxin exalted the efficiency of the initiation of infection to a constant level at any number higher than one of free p.f.u. initially present.

A brief reference is made to the mathematical treatment of these findings.

As a possible explanation of the phenomenon we propose that the polio receptor is a metastable pattern of macromolecules. The time of existence of a structure fully complementary to the attachment site(s) for virus may control the chance of reception and initiation of infection. The stabilization of a given conformational state for all or most (Na+, K+)-dependent ATPase molecules is believed to prolong the time of existence of the specific structural state required for reception and infection.

Nine 5-bromodeoxyuridine-induced temperature-sensitive (ts) mutants were isolated from stocks of a syncytial plaque type mutant (syn) itself derived from the non-syncytial wild-type (syn +) of the glasgow strain 17 of herpes simplex virus type 1. The nine ts mutants have been assigned to eight cistrons on the basis of different complementation tests.

Non-syncytial revertants isolated from the original ts syn mutants were used in reciprocal three-factor crosses of the type: tsX syn × tsY syn + and tsX syn + × tsY syn. With recombination data from these crosses a linkage map has been constructed. The provisional map which locates nine cistrons is linear and spans about 25 recombination units.

The regular occurrence of plaques with mixed syn/syn + morphology in the progeny from crosses is reported.

It has been shown by infectious centre assay of mixedly infected cells that ts mutants of HSV type 1 and type 2 are able to complement each other in many, but not all combinations. This complementation pattern has some unusual and unexpected features. Progeny tests on virus from infectious centres showed the presence of both parental types and also that of recombinants some of which have a novel phenotype. Successive progeny tests demonstrated that a substantial proportion of genomes retain the potential for segregating with respect to the ts and or the plaque morphology markers. The one recombinant so far tested behaved as intermediate between types 1 and 2 in neutralization tests.

A standard procedure for the isolation of virus specific double-stranded RNA from cowpea mosaic virus-infected Vigna leaves is described. The double stranded RNA is characterized by its buoyant density in caesium sulphate and its melting temperature (T m). The buoyant density and the T m are compared with those of other double-stranded RNA’s on basis of their guanine+cytosine content. The frequency distribution of the lengths of the molecules, determined by electron microscopy, indicate the occurrence of double-stranded RNA molecules specific for each of the two RNA’s of cowpea mosaic virus. During hybridization with labelled cowpea mosaic virus RNA, the virus RNA is specifically incorporated into the double-stranded structure.

Many viruses, in addition to the induction of c.p.e., produce profound biochemical alterations in the cell, particularly by the inhibition of cellular macromolecular synthesis (for review see Roizman & Spear, 1969). Reeve et al. (1971) have shown that the ability of different Newcastle disease virus (NDV) strains to inhibit cellular protein synthesis is related directly to their virulence for cells in vitro and for eggs and chickens in vivo.

Certain NDV strains can also inhibit cellular RNA synthesis, but the relationship of this property to the virulence of the infecting strain is not clear (Wheelock & Tamm, 1961; choltissek & Rott, 1965; Wilson, 1968). Moore, Lomniczi & Burke (1972) examined 13 strains of NDV but were unable to establish a relationship between virulence and the inhibition of host cell RNA synthesis.

Sindbis virus, a group A arbovirus, is composed of a nucleocapsid and a glycolipoprotein membrane (Pfefferkorn & Hunter, 1963a; Strauss et al. 1968; Schelle & Pfefferkorn, 1969). The membrane protein is specified by the virus genome (Pfefferkorn & Clifford, 1964; Strauss et al. 1968; Strauss, Burge & Darnell, 1969) whereas the phospholipids and carbohydrate are derived from the host of origin (Pfefferkorn & Hunter, 1963b; Burge & Strauss, 1969; Strauss, Burge & Darnell, 1970) during maturation by budding through the membranes of cytopathic vacuoles (CPV) or the plasma membranes of the infected cell (Grimley, Berezesky & Friedman, 1968; Hackett et al. 1968; Holmes, Wark & Warburton, 1969; Nakai, Shand & Howatson, 1969). We have studied the maturation of Sindbis virus with the use of ferritin-conjugated antibody.

Vaccinia virus has a complex structure. Thin sections of virus particles show three structural components: the outer envelope, the lateral bodies and a central structure, known as the ‘core’, which contains the virus DNA (Dales, 1963). Easterbrook (1966) showed that treatment with the nonionic detergent NP40, in the presence of 2-mercaptoethanol (2 ME) leads to the dissociation of the outer membrane and release of the core.

By polyacrylamide gel electrophoresis, Holowczak & Joklik (1967) identified at least 17 structural polypeptides, three of which were associated with the core. Katz & Moss (1970) showed that some of these polypeptide peaks could be further separated into two to three polypeptides, thus increasing the total number of resolved structural polypeptides to 22.

Much information regarding the association of the Epstein-Barr virus (EBV) with infectious mononucleosis, Burkitt’s lymphoma and nasopharyngeal carcinoma has been derived from serological test procedures, involving a number of distinct antigen-antibody systems related to EBV. These relationships have been recently reviewed (Henle & Henle, 1972).

There is evidence that the spleen is one of the main sites of interferon synthesis (Heineberg, Gold & Robbins, 1964; Kono & Ho, 1965; Fruitstone et al. 1966). Other studies have shown that the role of the spleen is more important in the production of interferon in response to intravenously administered endotoxin than in the synthesis of virus-induced interferon (De Somer & Billiau, 1966; Borecky & Lackovic, 1967; Ito et al. 1971). Effects of X-irradiation and antilymphocyte serum on the levels of circulating interferon prompted the conclusion that peripheral blood lymphocytes take part in myxovirus-induced interferon production (De Maeyer, De Maeyer-Guignard & Jullien, 1969; De Maeyer-Guignard & De Maeyer, 1971).

For practical purposes avian RNA tumour viruses have been divided into sarcoma and leukosis viruses. Sarcoma viruses have the ability to transform chick embryo fibroblasts in vitro and to induce fibro-sarcomas in vivo after a latent period of about 7 to 21 days. Leukosis viruses do not transform chick embryo fibroblasts, although they replicate in them, and in vivo most wild strains induce principally lymphoid leukosis after a latent period of over 90 days. They also frequently induce erythroid leukosis (erythroblastosis) and osteopetrosis, and occasionally other tumours.

Non-transforming mutants of sarcoma viruses, which have all the in vitro properties of leukosis viruses, have been produced experimentally by exposure to ultraviolet light (Toyoshima, Friis & Vogt, 1970), by γ-irradiation (Goldé, 1970) and by treatment with chemicals (Graf et al. 1971). These results suggest that leukosis viruses may originate from sarcoma viruses, perhaps by loss of genetic material either by mutation or by segregation of subgenomic components (Toyoshima et al. 1970; Graf et al. 1971; Martin & Duesberg, 1972). It has been suggested that non-transforming mutants arise spontaneously when cloned strains of Rous sarcoma viruses are grown (Vogt, 1971). However, regardless of whether such non-transforming viruses arise spontaneously or are produced experimentally, it is necessary to know whether they are oncogenic in vivo and behave like wild-type leukosis viruses.

Most preparations of infectious virus nucleic acids retain only about 0.1% or less of the infectivity of the original virus material. This is not necessarily the result of destruction of much of the intact nucleic acid during chemical procedures, but may be due to the insensitivity of the assay methods employed (Holland et al. 1961; Tovell & Colter, 1967). For the titration of infectious DNA obtained from polyoma virus, one of the methods in current use requires the direct plating of DNA in 0.55 m-NaCl on the monolayers of mouse embryo cells (Weil, 1961). This procedure has the disadvantage that the cell damage caused by the hypertonic NaCl renders accurate plaque counts difficult. A more recently described method (Warren & Thorne, 1968) avoids this complication by infecting monolayers in the presence of diethylaminoethyldextran (DEAE-dextran) in an isotonic medium. The infectious centres technique of Ellem & Colter (1960) also overcomes this difficulty but the application of this method to the assay of infective polyoma DNA was unsuccessful (Weil 1961). In this communication a method is described for the titration of polyoma DNA using an infectious centres technique, the sensitivity of which is enhanced by the addition of an appropriate dilution of dimethyl sulphoxide (DMSO).